Glutamine synthetase from E. coli is readily inactivated by mixed- function oxidation (MFO) wherein an activated oxygen species is thought to be generated by a protein bound transition metal such as Fe or Cu. Protection of glutamine synthetase against oxidative damage can be afforded by specific substrates (glu, ADP, etc.), metal cofactors (magnesium, manganese), and the transition state analog, L-methionine-SR-sulfoximine phosphate. This evidence strongly suggests that the location of this potential Cu or Fe binding site and activated oxygen generation is at the glutamine synthetase active site. To elucidate the structural origins of both MFO-dependent inactivation reactions and increased proteolytic susceptibility, absorption difference and second derivative rapid scan spectroscopies were employed to monitor changes in tyrosine and tryptophan microenvironments. Through the use of these spectral probes, the thermodynamic and kinetic alterations of a reversible thermal transition in glutamine synthetase were determined. MFO inactivation of glutamine synthetase results in decreased thermal stability, increased polarity surrounding tyrosine residues, and increased rates of proteolysis. The site specific nature of the MFO inactivation was further demonstrated by SDS-PAGE analysis of inactivated glutamine synthetase. Extensive exposure (greater than 4 hours) to nonenzymic mixed- function oxidation systems results in highly specific cleaved protein fragments whose quantity depends on the type of metal used (Fe or Cu), source of reducing equivalents (DTT, visible light + flavin, ascorbate) and the oxygen concentration.